As the human genome sequence project is completedthe vast amount of genomic information calls formethods that allow gene expression analysis on agenomic scale and speed up detection of significantsequence variations. The most suitable tools available forgene expressi

As the human genome sequence project is completed,
the vast amount of genomic information calls for
methods that allow gene expression analysis on a
genomic scale and speed up detection of significant
sequence variations. The most suitable tools available for
gene expression analysis are DNA hybridization arrays
that allow researchers to examine differences in expression
of hundreds or thousands of genes in parallel. That
is why DNA arrays, on a variety of platforms such as
macroarrays, microarrays or high-density oligonucleotide
arrays make their way into different areas of
functional genomics research. Arrays provide immobilized
gene-specific sequences (probes) on different
solid supports (e.g., nylon membranes, glass slides, or
silicon/ceramic chips) and are hybridized with labeled
targets that are copies of nucleic acids derived from
various biological sources.

This requires reliable and efficient tools for preparation
and labeling of targets: RNA needs to be extracted from
small amounts of precious sample materials, and labeled
either directly during cDNA preparation or via a linear
amplification (100 to 1000 fold) with T7 polymerase producing
labeled cRNA, or via polymerase chain reaction
(PCR)-based amplification methods. This step must be
highly reproducible and efficient to avoid differences in
hybridization caused by biased amplification of target
material.

Therefore, Roche Applied Science has developed a
range of DNA microarray analysis products for expression
profiling, suitable especially for very small amounts
of starting material or when highest sensitivity is
required. Each product is designed and tested in combination
with the others to cover the critical
workflow from
RNA isolation to preparation of labeled targets. After
each step, it is essential that residual impurities or
nucleases are removed from the reaction products
(single-stranded [ss] or double-stranded [ds] cDNA,
cRNA or PCR products). Therefore, we have developed
a nucleic acid purification kit that is universally
applicable to the whole range of potential samples.

Product Description

cDNA Synthesis System

The well-established cDNA Synthesis System provides a
convenient one-tube procedure for the synthesis of ds
cDNA, starting even with small amounts of total RNA
(120 g). Using the supplied Oligo ([dT24] T7 promoter)
primer, cDNA is synthesized containing a T7 RNA polymerase
promoter for the subsequent transcription reaction
with T7 RNA polymerase.

Microarray Target Purification Kit

The Microarray Target Purification Kit is specifically
designed for the purification of a variety of synthesized
nucleic acids in the target labeling and amplification
process, such as:

ss cDNA after 1st strand cDNA synthesis

ssDNA after cDNA labeling

ds cDNA after 2nd strand cDNA synthesis

PCR products

cRNA after in-vitro transcription

The kit will remove residual nucleotides, primers, proteins,
nucleases and other reaction components which
will otherwise interfere in the workflow for highly
sensitive microarray hybridization. The process does not
require nucleic acid precipitation, phenol/chloroform
extractions, or extensive handling of nucleic acids.

Microarray Target Synthesis Kit (T7)

The Microarray Target Synthesis Kit (T7) has been developed
as a robust, reliable and highly reproducible tool
for the preparation of large amounts of labeled cRNA
when starting with only 105 106 cells for target preparation.
Major features are:

acceptance of low amounts of cDNA (obtained
from 1 g total RNA)

short reaction time of only 2 3 hours (Figure 2)

high yields of cRNA in the range of 100 g

compatible to a broad spectrum of hapten or
fluorophore-labeled nucleotides

cost-effective labeling due to optimized nucleotide
concentrations and reaction conditions

Targets, synthesized and labeled with the Microarray
Target Synthesis Kit (T7) have been tested on various
commercially available DNA arrays. We have found that
it is sufficient to use only one labeled nucleotide to get
reliable results compared to double-labeling procedures
in the majority of experiments.

Very often sample material is available in such small
quantities (starting with less than 1,000 cells) that efficient
target preparation requires PCR-based amplification methods. Within the Microarray Target Synthesis Kit
(PCR), we have optimized a random primer-based PCR
amplification procedure which allows the efficient and
reproducible amplification of mRNA out of 50 ng total
RNA.

The resulting PCR product can then be labeled by different
methods, e.g., with the Microarray RNA Target
Synthesis Kit (T7) to obtain single stranded cRNA.

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